Combinations for the treatment of inflammatory skin disorders

ABSTRACT

The invention features methods and compositions for the treatment of inflammatory skin conditions.

BACKGROUND OF THE INVENTION

The invention relates to the treatment of inflammatory skin disorders.

Inflammatory skin disorders (e.g., psoriasis, inflammatory dermatoses,and atopic dermatoses) are characterized by dysregulation of the immunesystem and inappropriate activation of body's defenses, resulting indamage to healthy skin tissue. The damage results in redness, itching,swelling, and blistering of the skin.

Atopic dermatitis occurs in people who have a family history of asthma,allergic rhinitis, or atopic eczema, accompanied by chronic or recurrentdry, extremely itchy, inflamed lesions. The hypersensitivity or allergicreaction that occurs in the skin causes chronic inflammation. Persistentscratching often results in wounds that can become infected withbacteria or viruses. Staphylococcal infections are common in patientswith disorders of the skin. Atopic dermatitis affects approximately 10%of children, and in a small percentage of people the symptoms continueinto adulthood.

Psoriasis is a common chronic proliferative skin disease, affecting upto 2% of the population. One characteristic of psoriasis is a stronghyperproliferation of epidermal keratinocytes and an incompleteepidermal differentiation that leads to severe scaling of the affectedskin areas. This proliferative event is accompanied by an inflammationof the epidermis and dermis, with infiltrates of T-cells, neutrophils,and macrophages. Risk factors that indicate a higher chance of acquiringpsoriasis include: stress, infections, certain medications, immunologicfactors (e.g., HIV), and family history. There is some association witharthritis.

Treatment of inflammatory skin disorders generally involves reducingcontact with irritants (e.g., rough fibers, chemicals, make-up, etc.)and allergens (e.g., moulds, grass pollens, and animal dander), and theapplication of emollients to keep the skin moisturized. Topicalcorticosteroids have also been used as anti-inflammatory agents and totreat the symptoms of dermatoses. To date, however, no permanent cure ispossible, and there exists a need in the field for new and moreeffective agents that can be used to treat inflammatory skin disorders.

SUMMARY OF THE INVENTION

We have discovered that the combination of a steroid with aprostaglandin, a beta-adrenergic receptor ligand, an anti-mitotic agent,or a microtubule inhibitor brings about substantial suppression of TNFαlevels induced in white blood cells. TNFα is a major mediator ofinflammation. Specific blockade of TNFα using antibodies or solublereceptors is a potent treatment for patients having an inflammatory skindisease, such as psoriasis, inflammatory dermatitis, and atopicaldermatitis. Thus, this combination can be used to treat inflammatoryskin disorders. Moreover, based on the shared action among prostaglandinfamily members, beta-adrenergic receptor ligand family members,anti-mitotic agent family members, microtubule inhibitor family members,and steroid family members, any member of a family can be replaced byanother member of that family in the combination.

We have also discovered that the combination of a microtubule inhibitorwith an azole also provides substantial suppression of TNFα levelsinduced in white blood cells. Thus, this combination can similarly beused to treat inflammatory skin disorders. Based on the shared actionamong microtubule inhibitor family members and azole family members, onemember of a family can be replaced by another member of that family inthe combination.

Accordingly, the invention features a method for treating a patient whois diagnosed with, or is at risk for developing, an inflammatory skindisorder (e.g., psoriasis, inflammatory dermatitis, or atopicaldermatitis) by topically administering to the patient a prostaglandinand a steroid, in amounts that treat the patient. In one particularembodiment, the prostaglandin is alprostadil and the steroid isdiflorasone, prednisolone, or dexamethasone.

In another aspect, the invention also features another method fortreating a patient who is diagnosed with, or is at risk for developing,an inflammatory skin disorder by topically administering to the patienta beta-adrenergic receptor ligand and a steroid, in amounts that treatthe patient. One example features isoproterenol and diflorasone,prednisolone, or dexamethasone.

The invention features another method for treating a patient who isdiagnosed with, or is at risk for developing, an inflammatory skindisorder. In this method, an anti-mitotic agent and a steroid areadministered to the patient in amounts that treat the patient. Forexample podofilox (podophyllotoxin) can be used in combination with asteroid such as diflorasone, prednisolone, or dexamethasone.

Another method for treating a patient who is diagnosed with, or is atrisk for developing, an inflammatory skin disorder includes the step oftopically administering to the patient a microtubule inhibitor (e.g.,colchicine and vinblastine) and a steroid in amounts that treat thepatient. For example colchicine can be used in combination with asteroid such as diflorasone, prednisolone, or dexamethasone.

In yet another aspect, the invention, features a method for treating apatient who is diagnosed with, or is at risk for developing, aninflammatory skin disorder, by topically administering to the patient amicrotubule inhibitor (e.g., colchicine and a vinca alkaloid (e.g.,vinblastine)) and an azole (e.g., clotrimazole) in amounts that treatthe patient. For example vinblastine can be used in combination withclotrimazole.

In each of the foregoing methods, the two drugs can be topicallyadministered separately or together. If administered separately, thecompounds can be administered within 14 days of each other (e.g., within10 days, within five days, twenty-four hours, or one hour of eachother). Administration of each compound in the combination can occur 1to 10 times each day, desirably 1 to 8 times each day, more desirably 1to 6 times each day, most desirably 1 to 4 times each day, or asnecessary to alleviate symptoms.

The invention also features compositions containing one or more of thecompounds described above (i.e., a prostaglandin, a beta-adrenergicreceptor ligand, an anti-mitotic agent, or a microtubule inhibitor incombination with a steroid, and a microtubule inhibitor in combinationwith an azole). Particular compositions include alprostidil anddiflorasone; isoproterenol and prednisolone; podofilox anddexamethasone; colchicine and flumethasone; vinblastine andclotrimazole. Desirably, these compositions are formulated for topicaladministration and are in effective amounts for the treatment of aninflammatory skin disorder.

The invention also features a method of producing pharmaceuticalcompositions for treating an inflammatory skin disorder containing oneor more of the compounds described above (i.e., a prostaglandin, abeta-adrenergic receptor ligand, an anti-mitotic agent, or a microtubuleinhibitor in combination with a steroid, and a microtubule inhibitor incombination with an azole). The method is used to produce particularcompositions including alprostidil and diflorasone; isoproterenol andprednisolone; podofilox and dexamethasone; colchicine and flumethasone;vinblastine and clotrimazole. Desirably, the method is used to producecompositions formulated for topical administration and which incorporatethe compounds in effective amounts for the treatment of an inflammatoryskin disorder.

The specific amounts of the prostaglandin, the beta-adrenergic receptorligand, the anti-mitotic agent, the microtubule inhibitor, the steroid,and the azole administered depend on the specific combination ofcomponents and can be determined by one skilled in the art. Generally,when delivered by topical application, the prostaglandin,beta-adrenergic receptor ligand, anti-mitotic agent, and microtubuleinhibitor, are administered at a dose of 1 pg to 100 mg per day,desirably 1 pg to 75 mg per day, more desirably 1 pg to 50 mg per day,and most desirably 1 pg to 10 mg per day. The steroid is topicallyadministered at a total daily dosage of about 0.1 mg to 1500 mg per day,desirably about 0.1 mg to 200 mg per day, more desirably about 0.1 mg to100 mg per day, and most desirably 0.1 mg to 30 mg per day. Dosages ofup to 3000 mg per day may be necessary. The azole is topicallyadministered at a dosage of about 0.01 mg to 2000 mg per day, desirablyabout 0.01 mg to 800 mg per day, more desirably about 0.01 mg to 200 mgper day, and most desirably about 0.01 mg to 50 mg per day.

In several desired dose combinations, the ratio of prostaglandin (e.g.,alprostadil) to steroid (e.g., diflorasone) is desirably 10:1 to 20:1 byweight; the ratio of beta-adrenergic receptor ligand (e.g.,isoproterenol) to steroid (e.g., prednisolone) is desirably 10:1 to100:1 by weight; the ratio of anti-mitotic agent (e.g., podofilox) tosteroid (e.g., dexamethasone) is desirably 10:1 to 500:1 by weight; theratio of microtubule inhibitor (e.g., colchicine) to steroid (e.g.,flumethasone) is desirably 50:1 to 1000:1 by weight; the ratio ofmicrotubule inhibitor (e.g., vinblastine) to azole (e.g., clotrimazole)is desirably 2:1 to 1:2 by weight.

Compounds useful in the invention include those described herein in anyof their pharmaceutically acceptable forms, including isomers such asdiastereomers and enantiomers, salts, solvates, and polymorphs thereof,as well as racemic mixtures of the compounds described herein.

By an “amount sufficient to treat” is meant the amount of a compound, ina combination of the invention, required to reduce or prevent thesymptoms of an inflammatory skin disorder. A sufficient amount of activecompound(s) used to practice the present invention for therapeutictreatment of conditions caused by or contributed to by an inflammatoryskin disorder varies depending upon the manner of administration, theage, body weight, and general health of the patient. Ultimately, theattending physician or veterinarian will decide the appropriate amountand dosage regimen. Such amount is referred to as a sufficient amount.

By “anti-mitotic agent” is meant an agent that is capable of inhibitingmitosis. Exemplary anti-mitotic agents include, for example, podofilox,etoposide, teniposide, and griseofulvin.

By “azole” is meant any member of the class of anti-fungal compoundshaving a five-membered ring of three carbon atoms and two nitrogen atoms(e.g., the imidazoles) or two carbon atoms and three nitrogen atoms(e.g., triazoles), which are capable of inhibiting fungal growth. Acompound is considered “antifungal” if it inhibits growth of a speciesof fungus in vitro by at least 25%. Typically, azoles are administeredin dosages of greater than 200 mg per day when used as an antifungalagent. The azole can be selected from an imidazole or a triazole.Examples of exemplary imidazoles are sulconazole, miconazole,clotrimazole, oxiconazole, butocontazole, tioconazole, econazole, andketoconazole. Examples of exemplary triazoles are itraconazole,fluconazole, voriconazole, posaconazole, ravuconazole, and terconazole.

By “beta-adrenergic receptor ligand” is meant an agent that binds thebeta-adrenergic receptor in a sequence-specific manner. Exemplarybeta-adrenergic receptor ligands include agonists and antagonists.Exemplary beta-adrenergic receptor agonists include, for example,isoproterenol, dobutamine, metaproterenol, terbutaline, isoetharine,finoterol, formoterol, procaterol, ritodrine, salmeterol, bitolterol,pirbuterol, albuterol, levalbuterol, epinephrine, and ephedrine.Exemplary beta-adrenergic receptor antagonists include, for example,propanolol, nadolol, timolol, pindolol, labetolol, metoprolol, atenolol,esmolol, acebutolol, carvedilol, bopindolol, carteolol, oxprenolol,penbutolol, medroxalol, bucindolol, levobutolol, metipranolol,bisoprolol, nebivolol, betaxolol, celiprolol, solralol, and propafenone.

The term “inflammatory skin disorder” encompasses a variety ofconditions, including autoimmune diseases and proliferative skindiseases. Inflammatory skin disorders result in the damage of healthyskin tissue by an inflammatory process. Examples of inflammatory skindisorders include scleroderma, systemic lupus erythematosus, andinflammatory dermatoses. Inflammatory dermatoses include, for example,psoriasis, atopic dermatitis, non-specific dermatitis, lamellarichthyosis, epidemlolytic hyperkeratosis, premalignant keratosis, acne,and seborrheic dermatitis, pityriasis roseas, acute febrile neutrophilicdermatosis, eczema (e.g., asteatotic eczema, dyshidrotic eczema,vesicular palmoplantar eczema), balanitis circumscriptaplasmacellularis, balanoposthitis, Behcet disease, erythema annularecentrifugum, erythema dyschromicum perstans, erythema multiforme,granuloma annulare, lichen nitidus, lichen planus, lichen sclerosus etatrophicus, lichen simplex chronicus, lichen spinulosus, nummulardermatitis, pyoderma gangrenosum, sarcoidosis, subcorneal pustulardermatosis, urticaria, juvenile palmar-plantar dermatosis, keratosispilaris, acne fulminans, acrodermatitis enteropathica, infantileacropustulosis, mastocytomas, diffuse cutaneous mastocytosis, erythemamultiforme mino, erythema multiforme major, bullous dermatosis,alopecia, vitiligo, and transient acantholytic dermatosis.

By “reduce or prevent the symptoms of an inflammatory skin disorder” ismeant to lessen or inhibit pain, inflammation, itching, redness,swelling, blistering, dry skin, scaling, and lesions caused by orassociated with an inflammatory skin disorder.

By “microtubule inhibitor” is meant an agent that is capable ofaffecting the equilibrium between free tubulin dimers and assembledpolymers. Exemplary microtubule inhibitors include, for example,colchicine, vinca alkaloids (e.g., vinblastine, vincristine,vinorelbine, and vindesine), paclitaxel, and docetaxel.

By “prostaglandin” is meant a member of the lipid class of biochemicalsthat belongs to a subclass of lipids known as the eicosanoids, becauseof their structural similarities to the C-20 polyunsaturated fattyacids, the eicosaenoic acids. Exemplary prostaglandins includealprostidil, dinoprostone, misoprostil, prostaglandin E2, prostaglandinA1, prostaglandin A2, prostaglandin B1, prostaglandin B2, prostaglandinD2, prostaglandin F1α, prostaglandin F2α, prostaglandin I1,prostaglandin-ici 74205, prostaglandin F2β, 6-keto-prostaglandin F1α,prostaglandin E1 ethyl ester, prostaglandin E1 methyl ester,prostaglandin F2 methyl ester, arbaprostil, ornoprostil,13,14-dihydroprostaglandin F2α, and prostaglandin J.

By “steroid” is meant any naturally occurring or synthetic hormone thatcan be derived from cholesterol and is characterized by a hydrogenatedcyclopentanoperhydrophenanthrene ring system. Naturally occurringsteroids are generally produced by the adrenal cortex. Syntheticsteriods may be halogenated. Steroids may have corticoid,glucocorticoid, and/or mineralocorticoid activity. Examples of steroidsare algestone, 6-alpha-fluoroprednisolone, 6-alpha-methylprednisolone,6-alpha-methylprednisolone 21-acetate, 6-alpha-methylprednisolone21-hemisuccinate sodium salt, 6-alpha,9-alpha-difluoroprednisolone21-acetate 17-butyrate, amcinafal, beclomethasone, beclomethasonedipropionate, beclomethasone dipropionate monohydrate,6-beta-hydroxycortisol, betamethasone, betamethasone-17-valerate,budesonide, clobetasol, clobetasol propionate, clobetasone,clocoitolone, clocortol one pival ate, cortisone, cortisone acetate,cortodoxone, deflazacort, 21-deoxycortisol, deprodone, descinolone,desonide, desoximethasone, dexamethasone, dexamethasone-21-acetate,dichlorisone, diflorasone, diflorasone diacetate, diflucortolone,doxibetasol, fludrocortisone, flumethasone, flumethasone pivalate,flumoxonide, flunisolide, fluocinonide, fluocinolone acetonide,9-fluorocortisone, fluorohydroxyandrostenedione, fluorometholone,fluorometholone acetate, fluoxymesterone, flupredidene, fluprednisolone,flurandrenolide, formocortal, halcinonide, halometasone, halopredone,hyrcanoside, hydiocortisone, hydrocortisone acetate, hydrocortisonebutyrate, hydrocortisone cypionate, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, hydrocortisone probutate,hydrocortisone valerate, 6-hydroxydexamethasone, isoflupredone,isoflupredone acetate, isoprednidene, meclorisone, methylprednisolone,methylprednisolone acetate, methylprednisolone sodium succinate,paramethasone, paramethasone acetate, prednisolone, prednisoloneacetate, prednisolone metasulphobenzoate, prednisolone sodium phosphate,prednisolone tebutate, prednisolone-21-hemisuccinate free acid,prednisolone-21-acetate, prednisolone-21 (beta-D-glucuronide),prednisone, prednylidene, procinonide, tralonide, triamcinolone,triamcinolone acetonide, triamcinolone acetonide 21-palmitate,triamcinolone diacetate, triamcinolone hexacetonide, and wortmannin.Desirably, the corticosteroid is selected from cortisone, dexamethasone,hydrocortisone, methylprednisolone, prednisone, traimcinolone, anddiflorasone.

By “treating” is meant administering a pharmaceutical composition suchthat the symptoms of an inflammatory skin disorder are reduced orprevented. Administration may be to a patient already suffering from aninflammatory skin disorder to improve the patient's condition (i.e.,relieve pain, inflammation, itching, redness, swelling, blistering, dryskin, scaling, and lesions caused by ail inflammatory skin disorder, andhelp to maintain a patient's normal lifestyle) or to prevent theoccurrence or reoccurrence of an inflammatory skin disorder in apatient. By “patient” is meant any animal (e.g., a human).

The combinations described above for the treatment of an inflammatoryskin disorder allows for the administration of a low dose of eachcompound and less total active compound, thus providing similar efficacywith less toxicity, and reduced costs.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that the combination of a prostaglandin, abeta-adrenergic receptor ligand, an anti-mitotic agent, or a microtubuleinhibitor with a steroid, as well as the combination of a microtubuleinhibitor with an azole, has substantial TNFα suppressing activity onwhite blood cells. Concentrations that effectively suppress TNFαactivity are not unacceptably toxic to normal cells. Thus, combinationsof prostaglandins, beta-adrenergic receptor ligands, anti-mitoticagents, or microtubule inhibitors with steroids, as well as combinationsof microtubule inhibitors with azoles are useful for the treatment ofinflammatory skin disorders.

Therapy

Combination therapy according to the invention may be performed alone orin conjunction with another therapy and may be provided at home, thedoctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment generally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed. The duration of the combination therapy depends on the typeof disease or disorder being treated, the age and condition of thepatient, the stage and type of the patient's disease, and how thepatient responds to the treatment. Additionally, a person having agreater risk of developing an inflammatory skin disorder (e.g., a personwho is genetically predisposed or having a prior diagnosis of aninflammatory skin disorder) may receive prophylactic treatment toinhibit or delay the onset of symptoms.

An inflammatory skin disease is alleviated when there is a noticeabledecrease in a lesion of the skin, or a decrease in the presence ofitching, redness, swelling, blistering, or other manifestation of thedisease or condition. The alleviation of symptoms can occur without adecrease in residual redness, dilated blood vessels, hyper-pigmentation,or hypo-pigmentation. For the purposes of this invention, psoriasis isconsidered alleviated when a scale-free psoriasis lesion is noticeablydecreased in thickness.

The dosage, frequency, and mode of administration (e.g., gel, spray, orcream) of each component of the combination can be controlledindependently. For example, one compound may be administered topicallyby gel three times per day, while the second compound may beadministered topically by spray once per day. Combination therapy may begiven in on-and-off cycles that include rest periods so that thepatient's body has a chance to recovery from any as yet unforeseenside-effects. The compounds may also be formulated together such thatone administration delivers both compounds.

Formulation of Pharmaceutical Compositions

The compositions of the present invention are formulated for topicaladministration. Suitable formulations include gels, sprays, ointments,and creams. Administration of each compound of the combination may be byany suitable means that results in a concentration of the compound that,combined with the other compound, is effective. Each compound can beadmixed with a suitable carrier substance, and is generally present inan amount of 1-95% by weight of the total weight of the composition. Ifdesirable, the compounds can be formulated together.

The pharmaceutical compositions may be formulated for topical useaccording to conventional pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro,2000, Lippencott Williams & Wilkens, Philadelphia, Pa., and Encyclopediaof Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time period after administration,using controlled release topical formulations.

Therapeutic compositions suitable for topical application includeconventional anhydrous or aqueous preparations including ointments,lotions, creams, pastes, jellies, sprays, aerosols, and oils. Therepreparations can include oleaginous, aqueous, or emulsion-type bases.Optionally, topically applied formulations can be covered with anocclusive or semi-occlusive dressing.

Dosages

We have discovered that combinations of compounds can be used toeffectively treat inflammatory skin disorders. These combinationsinclude alprostadil and diflorasone; isoproterenol and prednisolone;podofilox and dexamethasone; colchicine and flumethasone; andvinblastine and clotrimazole. As is described herein, each of thesecompounds is a member of a larger family. Based on the shared actionamong family members, any member of a family can be replaced by anothermember of that family in the combination.

The dosage of each compound used in any given therapeutic method dependson several factors, including: the administration method, the conditionto be treated, the severity of the condition, whether the condition isto be treated or prevented, and the age, weight, and health of theperson to be treated. Additionally, pharmacogenomic (the effect ofgenotype on the pharmacokinetic, pharmacodynamic or efficacy profile ofa therapeutic) information about a particular patient may affect dosageused.

As is described above, the compound(s) are administered topically.Generally, when delivered by topical application, the prostaglandin,beta-adrenergic receptor ligand, anti-mitotic agent, and microtubuleinhibitor are administered at a dose of 1 pg to 100 mg per day,desirably 1 pg to 75 mg per day, more desirably 1 pg to 50 mg per day,and most desirably 1 pg mg to 10 mg per day (for a concentration rangeof between 0 and 500 μM, desirably 1 to 100 μM, more desirably 1 to 50μM, and most desirably 1 to 20 μM). The steroid is topicallyadministered at a total daily dosage of about 0.1 mg to 1500 mg per day,desirably about 0.1 mg to 200 mg per day, more desirably about 0.1 mg to100 mg per day, and most desirably 0.1 mg to 30 mg per day. Dosages ofup to 3000 mg per day may be necessary. The azole is topicallyadministered at a dosage of about 0.01 mg to 2000 mg per day, desirablyabout 0.01 mg to 800 mg per day, more desirably about 0.01 mg to 200 mgper day, and most desirably about 0.01 mg to 50 mg per day.

Combination effects are seen at all ratios tested, however, the besteffects are seen with particular ratios. Desired ratios for thecombinations are as follows: the ratio of prostaglandin (e.g.,alprostadil) to steroid (e.g., diflorasone) is desirably 10:1 to 20:1 byweight; the ratio of beta-adrenergic receptor ligand (e.g.,isoproterenol) to steroid (e.g., prednisolone) is desirably 10:1 to100:1 by weight; the ratio of anti-mitotic agent (e.g., podofilox) tosteroid (e.g., dexamethasone) is desirably 10:1 to 500:1 by weight; theratio of microtubule inhibitor (e.g., colchicine) to steroid (e.g.,flumethasone) is desirably 50:1 to 1000:1 by weight; and the ratio ofmicrotubule inhibitor (e.g., vinblastine) to azole (e.g., clotrimazole)is desirably 2:1 to 1:2 by weight.

Administration can be one to four times daily for one day to one year,and may even be for the life of the patient. Chronic, long-termadministration will be indicated in many cases.

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

EXAMPLE 1 Assay for TNFα Suppressing Activity

The compound dilution matrix was assayed using a TNFα ELISA method.Briefly, a 100 μl suspension of diluted human white blood cellscontained within each well of a polystyrene 384-well plate (NalgeNunc)was stimulated to secrete TNFα by treatment with a final concentrationof 10 ng/nl phorbol 12-myristate 13-acetate (Sigma) and 750 ng/mlionomycin (Sigma). Various concentrations of each test compound wereadded at the time of stimulation. After 16-18 hours of incubation at 37°C. in a humidified incubator, the plate was centrifuged and thesupernatant transferred to a white opaque polystyrene 384 well plate(NalgeNunc, Maxisorb) coated with an anti-TNFα antibody (PharMingen,#18631D). After a two-hour incubation, the plate was washed (TecanPowerWasher 384) with phosphate buffered saline (PBS) containing 0.1%Tween 20 (polyoxyethylene sorbitan monolaurate) and incubated for anadditional one hour with another anti-TNFα antibody that was biotinlabeled (PharMingen, 18642D) and horseradish peroxidase (HRP) coupled tostrepavidin (PharMingen, #13047E).). After the plate was washed with0.1% Tween 20/PBS, an HRP-luminescent substrate was added to each welland light intensity measured using a LJL Analyst plate luminometer. Setsof control wells contained a serial dilution of Cyclosporin A (Sigma)starting at a final concentration of 0.5 μg/ml.

EXAMPLE 2 Preparation of Combinations of Compounds

Stock solutions containing a prostaglandin, a beta-adrenergic receptorligand, an anti-mitotic agent, a microtubule inhibitor, a steroid, or anazole were made in dimethylsulfoxide (DMSO) at a final concentration ofbetween 0 and 20 μM. Master plates were prepared to contain dilutions ofthe stock solutions of the compounds described above. Master plates weresealed and stored at −20° C. until ready for use.

The final pairwise combination plates were generated by transferringstock solution from the specific master plates to a dilution platecontaining 100 μl of media (RPMI; Gibco BRL, #11875-085), 10% FetalBovine Serum (Gibco BRL, #25140-097), 2% Penicillin/Streptomycin (GibcoBRL, #15140-122)) using the TomTec Quadra Plus liquid handler. Thisdilution plate was then mixed and a 10 μl aliquot transferred to thefinal assay plate, which had been pre-filled with 40 μl/well RPMI mediacontaining the appropriate stimulant to activate TNFα secretion (seebelow).

EXAMPLE 3 Testing of Combinations for TNFα Suppressing Activity

Pair combinations were tested for the ability to suppress TNF secretionfrom stimulated white blood cells. TNF suppressing activity wasinvestigated using low doses of alprostadil with diflorasone (see Table1); isoproterenol with prednisolone (see Table 2), podofilox withdexamethasone (see Table 3), colchicine with flumethasone (see Table 4),and vinblastine with clotrimazole (see Table 5) significantly increasedthe suppression of TNF secretion from stimulated white blood cells.TABLE 1 Diflorasone (μM) 0.12 0.06 0.03 0.015 0.0075 0.0037 0.00180.0009 0.0004 0 Alprostadil 1 72.62 72.67 70.77 70.29 67.23 63.22 59.1153.39 52.08 37.49 (μM) 0.5 68.73 67.05 68.08 69.60 66.32 60.6 56.4151.82 45.67 34.57 0.25 69.15 68.18 68.83 67.08 62.40 59.85 52.13 50.2844.76 33.03 0.13 65.79 66.39 65.69 65.59 61.22 55.74 49.80 45.55 46.0936.80 0.06 65.39 63.34 63.46 62.61 56.36 50.04 48.63 47.49 39.66 34.110.03 61.84 62.71 62.43 60.49 56.76 50.03 47.30 40.78 37.18 30.66 0.0260.27 61.21 59.78 57.56 54.35 49.38 44.12 41.64 38.20 26.54 0.01 59.9957.18 55.74 53.33 48.4 45.81 37.46 35.22 29.74 19.25 0.005 55.36 52.2256.57 53.13 46.53 38.45 35.02 33.69 29.39 14.53 0 42.79 40.28 42.1135.75 32.96 28.52 15.85 12.86 5.28 −2.83

TABLE 2 Prednisolone (μM) 0.1 0.05 0.03 0.015 0.075 0.038 0.019 0.0090.004 0 Isoproteronol 2 56.55 49.98 40.08 34.84 36.5 29.63 28.04 22.0522.79 18.92 (μM) 1 51.69 47.29 42.75 39.21 34.56 34.27 32.59 26.86 26.9923.42 0.5 49.63 39.36 41.34 30.87 30.26 24.39 25.72 20.39 12.93 13.880.25 48.74 46.84 34.12 27.91 28.02 23.50 19.96 19.90 17.09 8.58 0.1348.99 38.70 29.84 28.60 17.99 24.11 13.79 21.75 13.88 6.27 0.06 48.3635.48 31.05 23.60 21.35 20.60 16.22 14.38 11.04 10.52 0.03 41.27 30.5131.58 18.18 22.08 23.46 16.56 21.76 15.26 10.44 0.02 44.75 35.06 30.0324.33 21.31 19.11 16.18 17.35 9.50 7.24 0.01 40.89 29.10 26.05 24.4320.71 14.04 14.35 10.37 7.26 5.35 0 40.87 32.38 22.89 21.29 16.24 18.7119.09 12.20 6.05 0

TABLE 3 Dexamethasone (μM) 0.1 0.05 0.03 0.015 0.0075 0.0038 0.00190.0009 0.0005 0 Podofilox 2.41 66.35 65.60 58.89 58.67 52.35 49.36 43.6343.38 41.73 34.00 (μM) 1.21 68.40 64.47 60.49 55.40 53.92 49.33 45.4242.70 42.58 32.73 0.6 66.95 65.55 61.60 58.54 51.04 48.45 44.84 45.0843.96 38.66 0.3 66.27 63.17 62.02 55.89 53.76 49.49 47.58 44.23 42.2339.48 0.15 65.27 62.18 58.20 52.39 55.23 50.34 48.43 44.68 43.07 41.150.08 61.43 56.80 59.20 50.44 49.69 43.20 44.08 44.02 36.17 29.63 0.0442.29 39.13 45.60 29.40 30.17 36.57 15.82 14.21 7.54 5.91 0.02 43.4239.18 33.28 24.92 19.40 32.89 7.47 5.22 5.82 −3.32 0.01 42.18 38.8331.75 29.10 18.61 10.76 8.01 4.78 −2.77 0.02 0 44.50 35.42 32.43 27.3622.18 7.26 11.32 6.4 4.97 −1.21

TABLE 4 Flumethasone (μM) 0.04 0.02 0.01 0.0050 0.0025 0.0013 0.00060.0003 0.0002 0 Colchicine 2.5 72.68 70.14 68.32 63.85 59.22 52.28 49.5847.18 45.96 41.21 (μM) 1.25 69.35 66.52 60.86 57.04 53.25 45.46 43.7741.35 39.18 34.11 0.63 63.94 60.64 56.34 53.06 46.59 40.73 36.21 34.0531.39 28.69 0.31 57.81 54.31 52.24 45.42 39.92 32.60 31.66 28.96 23.7823.49 0.16 56.29 51.01 48.73 41.14 36.60 31.01 28.89 17.43 19.00 14.330.08 50.62 46.79 40.22 31.05 25.80 22.33 20.98 12.76 9.38 7.94 0.0453.98 48.14 42.82 38.49 31.77 26.54 21.04 17.00 10.56 6.56 0.02 47.7746.68 42.12 38.69 27.81 21.19 11.01 11.11 4.58 4.23 0.01 45.53 45.7136.8 26.64 24.14 14.34 10.60 6.02 1.87 0.75 0 49.24 41.29 36.52 29.5621.52 11.17 12.52 5.10 −1.75 −5.53

TABLE 5 Clotrimazole (μM) 2 1 0.5 0.25 0.13 0.06 0.03 0.02 0.01 0Vinbalstine 1.1 80.19 77.75 72.74 68.28 61.34 59.49 53.71 59.72 57.7048.98 (μM) 0.55 79.63 74.72 70.32 64.4 61.23 60.63 55.98 54.76 50.4351.76 0.28 77.35 68.40 66.67 59.76 56.88 54.22 55.26 35.45 38.94 44.010.14 74.28 68.48 60.16 60.50 55.49 50.27 49.76 47.45 49.19 45.63 0.0766.63 62.30 57.65 47.67 43.64 45.01 47.20 42.94 37.66 43.87 0.03 69.3760.02 55.76 43.95 46.07 44.77 46.88 43.79 39.77 34.21 0.02 67.38 55.9052.96 45.93 47.04 29.34 38.80 30.67 34.30 27.47 0.01 61.94 57.29 55.6043.23 42.45 37.81 29.81 32.02 28.98 38.13 0 57.95 49.45 36.59 37.0331.31 13.55 28.18 24.66 15.81 11.38 0 51.68 46.86 37.27 27.77 17.5710.18 1.52 −2.42 −0.69 0

OTHER EMBODIMENTS

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in cellularand molecular biology, pharmacology, immunology, or related fields areintended to be within the scope of the invention.

1. A method for treating a patient diagnosed with or at risk fordeveloping an inflammatory skin disorder, said method comprisingadministering to said patient a prostaglandin and a steroid, whereinsaid prostaglandin and said steroid are topically administeredsimultaneously or within fourteen days of each other, in amountssufficient to treat said patient.
 2. The method of claim 1, wherein saidprostaglandin is alprostidil, misoprostil, dinoprostone, prostaglandinE2, prostaglandin A1, prostaglandin A2, prostaglandin B1, prostaglandinB2, prostaglandin D2, prostaglandin F1α, prostaglandin F2α,prostaglandin I1, prostaglandin-ici 74205, prostaglandin F2β,6-keto-prostaglandin F1α, prostaglandin E1 ethyl ester, prostaglandin E1methyl ester, prostaglandin F2 methyl ester, arbaprostil, omoprostil,13,14-dihydroprostaglandin F2α, or prostaglandin J.
 3. The method ofclaim 2, wherein said prostaglandin is alprostidil.
 4. The method ofclaim 1, wherein said prostaglandin is alprostidil and said steroid isdiflorasone.
 5. A method for treating a patient diagnosed with or atrisk for developing an inflammatory skin disorder, said methodcomprising administering to said patient a beta-adrenergic receptorligand and a steroid, wherein said beta-adrenergic receptor ligand andsaid steroid are topically administered simultaneously or withinfourteen days of each other, in amounts sufficient to treat saidpatient.
 6. The method of claim 5, wherein said beta-adrenergic receptorligand is isoproterenol, dobutamine, metaproterenol, terbutaline,isoetharine, finoterol, formoterol, procaterol, ritodrine, salmeterol,bitolterol, pirbuterol, albuterol, levalbuterol, epinephrine, ephedrine,propanolol, nadolol, timolol, pindolol, labetolol, metoprolol, atenolol,esmolol, acebutolol, carvedilol, bopindolol, carteolol, oxprenolol,penbutolol, medroxalol, bucindolol, levobutolol, metipranolol,bisoprolol, nebivolol, betaxolol, celiprolol, solralol, or propafenone.7. The method of claim 6, wherein said beta-adrenergic receptor ligandis isoproterenol.
 8. The method of claim 5, wherein said beta-adrenergicreceptor ligand is isoproterenol and said steroid is prednisolone.
 9. Amethod for treating a patient diagnosed with or at risk for developingan inflammatory skin disorder, said method comprising administering tosaid patient an anti-mitotic agent and a steroid, wherein saidanti-mitotic agent and said steroid are topically administeredsimultaneously or within fourteen days of each other, in amountssufficient to treat said patient.
 10. The method of claim 9, whereinsaid anti-mitotic agent is podofilox, etoposide, teniposide, orgriseofulvin.
 11. The method of claim 10, wherein said antimitotic agentis podofilox.
 12. The method of claim 9, wherein said anti-mitotic agentis podofilox and said steroid is dexamethasone.
 13. A method fortreating a patient diagnosed with or at risk for developing aninflammatory skin disorder, said method comprising administering to saidpatient a microtubule inhibitor and a steroid, wherein said microtubuleinhibitor and said steroid are topically administered simultaneously orwithin fourteen days of each other, in amounts sufficient to treat saidpatient.
 14. The method of claim 13, wherein said microtubule inhibitoris an alkaloid, paclitaxel, or docetaxel.
 15. The method of claim 14,wherein said alkaloid is colchicine or a vinca alkaloid.
 16. The methodof claim 15, wherein said vinca alkaloid is vinblastine, vincristine,vinorelbine, or vindesine.
 17. The method of claim 13, wherein saidmicrotubule inhibitor is colchicine and said steroid is dexamethasone.18. The method of claim 1, wherein said steroid is dexamethasone,diflorasone, flumethasone, or prednisolone. 19-26. (canceled)
 27. Themethod of claim 1, wherein said combination is administered within tendays of each other.
 28. The method of claim 27, wherein said combinationis administered within five days of each other.
 29. The method of claim28, wherein said combination is administered within twenty-four hours ofeach other.
 30. The method of claims 1, wherein said inflammatory skindisorder is psoriasis, inflammatory dermatosis, or atopic dermatosis.31. A pharmaceutical composition comprising a prostaglandin, a steroid,and a pharmaceutically acceptable carrier, wherein said prostaglandinand said steroid are present in amounts that, when topicallyadministered to a patient, reduce or prevent the symptoms of aninflammatory skin disorder. 32-40. (canceled)
 41. A method of producinga pharmaceutical composition for treating an inflammatory skin disorder,said method comprising admixing a prostaglandin, a steroid, and apharmaceutically acceptable carrier, wherein said prostaglandin and saidsteroid are present in amounts that, when topically administered to apatient, reduce or prevent the symptoms of an inflammatory skindisorder. 42-81. (canceled)